The overall goal of this proposal is to exploit our understanding of the Ral signaling cascade to reveal how it contributes to cancer in both tumor cells and adjacent stromal fibroblasts. RalA and RalB are members of the Ras superfamily that become activated by a distinct set of guanine nucleotide exchange factors, like RalGDS, in response to a variety of extracellular signals. Once activated, Ral proteins influence a unique set of downstream signaling molecules that regulate multiple cellular processes including vesicle trafficking, apoptosis, cell migration and cell proliferation. A key property of the Ral signaling cascade is that it is stimulated by Ras proteins. In many studies, including those involving RalGDS knockout mice, the Ral signaling cascade supports Ras-induced oncogenic transformation, however the mechanisms involved are poorly understood. Thus, there is intense interest in revealing how the Ral signaling cascade contributes to cancer. In order to understand how Ral GTPases function in squamous carcinoma of the skin, where activated Ras is often an important component, we used a bioengineered tissue model of human skin that allows us to manipulate the Ral signaling cascade in both epithelial and stromal compartments. We found that RalA plays a cell-type dependent role in Ras-mediated squamous cell carcinoma. In keratinocytes of the epithelium, RalA inhibits, rather than supports tumorigenesis, since suppression of RalA expression enhances tumor progression at least in part by promoting cell invasiveness, through its effector protein the exocyst subunit Exo84 and decreased E-cadherin stability. Moreover, tumor progression in this model system is associated with, and requires, down-regulation of RalA levels. RalB knock-down complements the effects of RalA inhibition by enhancing keratinocyte proliferation. Specific Aim 1 will elucidate how RalA and RalB play these surprising tumor-suppressing activities, and reveal how tumor progression down-regulates RalA levels in cells to allow tumor progression. In fibroblasts of the dermis, RalA has the opposite function. It supports tumorigenesis, since RalA knock-down in these cells blocks the invasive properties of adjacent epithelial cells. Specific Aim 2 will reveal the mechanism behind this striking phenomenon and test the hypothesis that at least part of the tumor- resistant phenotype of RalGDS knockout mice is due to the loss of this protein in dermal fibroblasts. Finally, we will test the exciting possibility that the components of a RalA signaling cascade in genetically stable fibroblasts represent new drug targets to block the formation of not only skin squamous carcinoma but also breast adenocarcinoma. PUBLIC HEALTH RELEVANCE: There is abundant evidence that the tissue microenvironment has a major effect on carcinoma progression. As such, the mechanisms underlying this phenomenon, and how to exploit them to develop new strategies to block tumor progression are aggressively being pursued. In this proposal, we address these issues by studying the role of Ral GTPases in both keratinocytes and adjacent stromal fibroblasts using a bioengineered human tissue model of skin squamous cell carcinoma, as well as an animal model of this disease. It is anticipated that new strategies to block tumor development by targeting the Ral GTPase signaling cascade in epithelial cells and/or stromal fibroblasts will emerge from the result of experiments described in this proposal.